Kinetoplastid parasites are protozoa that cause devastating diseases in much of the tropical and subtropical world. They are the causative agents of visceral and cutaneous leishmaniasis, sleeping sickness and Chagas disease. These microorganisms are characterized by a unique form of mitochondrial DNA called kinetoplast DNA or kDNA. This DNA consists of several thousand minicircles and about 25 maxicircles, all of which are interlocked into a DNA network. Our long term goal is to reveal the unique molecular and biochemical mechanisms involved in the replication of kDNA and its regulation. These studies use the trypanosomatid Crithidia fasciculata as a model kinetoplastid because of the ability to readily carryout a wide range of molecular genetic and biochemical techniques and the extensive knowledge of kDNA replication mechanisms in this organism. The proposed studies address two areas concerning kDNA replication and its regulation. First, a novel mechanism of gene regulation discovered in previous studies will be investigated to identify components of the regulatory mechanism. A protein termed the cycling element binding protein (CEBP) binds to a consensus octamer sequence in the 5'UTR of the mRNAs of several DNA replication genes. These octamer sequences are required for periodic accumulation of each of the mRNAs. We will purify CEBP and clone the gene(s) encoding CEBP. Biochemical and molecular genetic approaches will be used to investigate the role of CEBP in the regulation of specific mRNA levels during the cell cycle. Previous work has shown that newly replicated minicircles have gaps and remnants of RNA primers in the newly synthesized strands that remain until all minicircles have been replicated and until just prior to network division. We will study the roles of several proteins implicated in the late stages of kDNA replication and also possibly in division of the kDNA network. These include a ribonuclease H, an enzyme that degrades the RNA strand of RNA-DNA hybrids, a structure-specific endonuclease and a protein associated with the kinetoplast type II DNA topoisomerase.